Citron Kinase Controls a Molecular Network Required for Midbody Formation in Cytokinesis
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Citron kinase controls a molecular network required for midbody formation in cytokinesis Zuni I. Bassia, Morgane Audusseaua, Maria Giovanna Riparbellib, Giuliano Callainib, and Pier Paolo D’Avinoa,1 aDepartment of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom; and bDepartment of Evolutionary Biology, University of Siena, 53100 Siena, Italy Edited by R. Scott Hawley, Stowers Institute for Medical Research, Kansas City, MO, and approved April 30, 2013 (received for review January 22, 2013) Cytokinesis partitions cytoplasmic and genomic materials at the during cytokinesis, but the exact function of this interaction is still end of cell division. Failure in this process causes polyploidy, which obscure. CIT-K was originally identified as a RhoA effector that in turn can generate chromosomal instability, a hallmark of many could regulate myosin contractility by phosphorylating the myosin cancers. Successful cytokinesis requires cooperative interaction be- regulatory light chain (10, 11). However, CIT-K and its Drosophila tween contractile ring and central spindle components, but how this ortholog, Sticky (Sti), are not required for furrowing (12–16), and cooperation is established is poorly understood. Here we show that evidence in both Drosophila and human cells has challenged the Sticky (Sti), the Drosophila ortholog of the contractile ring com- original model by showing that CIT-K is required for proper ponent Citron kinase (CIT-K), interacts directly with two kinesins, RhoA localization at the cleavage site during late cytokinesis, thus Nebbish [the fly counterpart of human kinesin family member 14 behaving more like a RhoA regulator than an effector (14, 17). (KIF14)] and Pavarotti [the Drosophila ortholog of human mitotic Moreover, Sti is not required for myosin regulatory light chain kinesin-like protein 1 (MKLP1)], and that in turn these kinesins in- phosphorylation and associates with actomyosin filaments to lo- teract with each other and with another central spindle protein, calize to the cleavage furrow (17). This indicates that CIT-K acts Fascetto [the fly ortholog of protein regulator of cytokinesis 1 as an important regulator of late events during cytokinesis, but the (PRC1)]. Sti recruits Nebbish to the cleavage furrow, and both pro- molecular details are still missing. Here we show that Sti directly teins are required for midbody formation and proper localization of binds to two kinesins, Pavarotti [Pav, the Drosophila ortholog of Pavarotti and Fascetto. These functions require Sti kinase activity, human mitotic kinesin-like protein 1 (MKLP1), the other cen- indicating that Sti plays both structural and regulatory roles in mid- tralspindlin component] (18) and Nebbish (Neb, the fly counter- CELL BIOLOGY body formation. Finally, we show that CIT-K’s role in midbody for- part of human KIF14) (19–21), which in turn interact with each mation is conserved in human cells. Our findings indicate that CIT-K other and with another central spindle component, Fascetto [Feo, is likely to act at the top of the midbody-formation hierarchy by the fly ortholog of protein regulator of cytokinesis 1 (PRC1)] (22). connecting and regulating a molecular network of contractile ring Furthermore, we show that Sti depletion affects MB formation components and microtubule-associated proteins. and localization of Neb, Pav, and Feo, and that Sti kinase activity is necessary for these functions. We also found that Neb depletion phenocopied sti RNAi MB defects. Finally, we report that CIT-K ytokinesis requires the coordinated action of many proteins fi Cthat control sequential events. First, cells position the performs similar roles in human cells. Our ndings, together with cleavage plane through signals generated by the spindle micro- those of previous studies (14, 17), indicate that Sti/CIT-K acts as tubules, which, during anaphase, reorganize into an array of a key regulator of MB formation in late cytokinesis through its antiparallel and interdigitating microtubules known as the cen- association with a network made up of contractile ring com- tral spindle. Central spindle and astral microtubules promote ponents (anillin, actin, myosin, and RhoA) and at least three ingression of the cleavage furrow that bisects the dividing cell microtubule-associated proteins (MAPs; Neb, Pav, and Feo) (1). Furrow ingression, driven by assembly and constriction of necessary for central spindle organization. the actomyosin contractile ring, compacts the central spindle to Results form a structure known as the midbody (MB), which contains at its center an electron-dense structure, the MB ring or Flemming N-Terminal Region of the Sti Coiled-Coil Domain Interacts with the fi body, which is important for abscission. Kinesins Pav and Neb. We previously identi ed a region in the Sti “ ” Cleavage furrow ingression and MB formation require coop- coiled-coil domain, dubbed StiCC1, which unexpectedly lo- A eration between contractile ring and central spindle factors, but calized to the central spindle midzone (Fig. 1 and see Fig. S2) how this is established is poorly understood (2, 3). Some inter- (17). This localization differs from that of endogenous Sti, which actions between the components of these two structures have instead accumulates at the cleavage furrow (12, 17). As StiCC1 is been described. The contractile ring component anillin asso- not necessary for Sti localization to the cleavage furrow (17), we ciates with two central spindle proteins: the RhoGEF epithelial reasoned that StiCC1 localization to the midzone might reflect cell transforming sequence 2 (ECT2), responsible for RhoA an interaction with one or more MAPs. To identify these MAPs, activation at the cleavage site, and the RacGAP component of we tagged both StiCC1 and full-length Sti (StiFL) with two IgG the centralspindlin complex (4–6), a key regulator of multiple binding motifs of protein A (PtA) and isolated proteins that events during cytokinesis, including furrowing, central spindle interact with these two baits by affinity purification coupled with assembly, and MB formation and stability (7). However, anillin mass spectrometry (23). This approach led to the identification depletion does not affect localization of either protein to the of two kinesins, Pav and Neb (Table S1). In vivo pull-down assay central spindle midzone (4–6), suggesting that these interactions confirmed that PtA::StiCC1 formed a complex with both kinesins, do not play a major structural role but, rather, establish a platform necessary to maximize the efficiency of signaling pathways re- quired for cleavage furrow ingression (8). Another contractile ring Author contributions: P.P.D. designed research; Z.I.B., M.A., M.G.R., G.C., and P.P.D. per- component, the serine/threonine kinase Citron kinase (CIT-K), formed research; Z.I.B. and P.P.D. analyzed data; and P.P.D. wrote the paper. associates with the central spindle kinesin family member 14 The authors declare no conflict of interest. (KIF14), and these two proteins depend on each other for proper This article is a PNAS Direct Submission. localization during cytokinesis (9). This led to the proposal that 1To whom correspondence should be addressed. E-mail: [email protected]. KIF14 could direct CIT-K localization to the cleavage site at This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. furrowing onset and that the two proteins could act together 1073/pnas.1301328110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1301328110 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 Fig. 1. Sti interacts with Neb and Pav via its CC1 region. (A) Schematic diagrams illustrating the protein domains of Sti, Neb, and Pav and the posi- tions of the different fragments used for the in vitro pull-down assays. (B) GST::StiCC1 and GST alone were incubated with in vitro translated and radio- labeled Neb, Pav, and Feo polypeptides (Right)and then pulled down using glutathione beads. (C)The GST::Neb proteins indicated at the top and GST alone were incubated with the in vitro translated and radiolabeled StiCC1, Pav, and Feo polypeptides indicated at the right, and then pulled down using glutathione beads. (D) The GST::Pav proteins in- dicated at the top and GST alone were incubated with in vitro translated and radiolabeled StiCC1, Neb1–740, and Feo polypeptides and then pulled down using glutathione beads. (E) GST::StiCC1 and GST alone were incubated with a mixture contain- ing both Neb741–1121 and Pav461–685 translated and radiolabeled in vitro and then pulled down using glutathione beads. (F) GST::StiCC1 was incubated with an in vitro translated and radiolabeled Pav461–685 polypeptide and increasing concentrations of GST:: Neb741–1121. The mixtures were then pulled down using glutathione beads. In all panels, the Ponceau staining of the protein loading is shown in the lower part and the numbers on the left indicate the sizes, in kilodaltons, of the molecular mass marker. although only in the presence of a cyclin-dependent kinase 1 (Cdk1) Neb in vitro. First, GST::StiCC1 could simultaneously pull down inhibitor (Fig. S1), thus suggesting that this ternary complex as- both Neb741–1121 and Pav461–685 (Fig. 1E). Second, the binding of sembled preferentially after anaphase onset. Consistent with these GST::StiCC1 to radiolabeled Pav461–685 was unaffected by the findings, we found that Neb depletion prevented accumulation of presence of increasing concentrations of GST-Neb741–1121, which F StiCC1 at the midzone (Fig. S2). We could not test whether Pav bindstoStiCC1butnottoPav461–685 (Fig. 1 ). Thus, Pav and Neb was necessary for StiCC1 localization to the midzone because Pav do not seem to compete for their binding to StiCC1. In sum, our depletion inhibits furrow ingression (24). findings indicate that Sti, Neb, Pav, and Feo form a complex in To assess whether StiCC1 could directly bind to Pav and Neb, vivo in which the StiCC1 region binds to Pav and Neb, and in turn we purified a StiCC1 fragment tagged with glutathione S-trans- these two kinesins interact directly with each other and with Feo.